Malaria, dengue and dengue haemorrhagic fever, WNV and other encephalites, human African trypanosomiasis (HAT), human filariasis, dog heartworm and other pathogens important to animals are on the increase. Although vector control methods are available to interrupt transmission of these diseases, their effectiveness has been limited by logistic problems, development of resistance to insecticides, regulatory restrictions related to environmental concerns and high cost. Novel, sustainable approaches to control are urgently needed.
Recent molecular advances in the understanding of vector genetics and vector-parasite/virus relationships have provided novel tools for the study of disease transmission. Among these are germ-line transformation of mosquitoes, detailed genetic and physical maps, molecular genetic markers for the identification of cryptic species, detection of pathogens in vectors, gene flow studies, detection of insecticide resistance, and the complete genome of Anopheles gambiae as well as collections of expressed sequence tags (ESTs) from a variety of disease vectors. However, much work remains to be done to identify, at the molecular level, the role of insect vectors in disease transmission, and the mechanisms for interfering with vector competence.
Insect vectors of disease constitute a major threat to human and animal health. Malaria, the most prevalent of the mosquito-born diseases, afflicts 300-400 million people annually, of which about 1% die from the disease. Malaria and other vector-borne diseases are increasing in the world today for a variety of reasons, including vector resistance to pesticides, parasite resistance to drugs, more movement by people, climatic changes, etc. Warm areas of the United States, such as Arizona, with its proximity to the reservoirs of disease in Central America, are especially vulnerable to spread of vector-borne diseases. Attempts to lessen the impact of malaria and the other insect-born diseases require extensive knowledge of the vector insect, of the causative agent, and of the interaction between vector and parasite or vector and virus.
The primary indirect effect of medical and veterinary insects is disease transmission. Indeed, disease transmission is more important than any other effect produced by medical and veterinary pests. Underlying the relationship of arthropods to disease requires consideration of many concepts and much terminology.
Organisms that produce disease are called pathogens and disease itself is a stress condition produced by the effects of a pathogen on a susceptible host. Arthropods capable of transmitting pathogens are called vectors. Some diseases may depend on only a single host and a vector; however, other diseases may include multiple host species, and even multiple vectors. In many of these instances, an organism that maintains the infective agent (the pathogen source) when active transmission does not occur is termed a reservoir. For example, the reservoir for malaria is human populations, with transmission occurring when a mosquito feeds on an infected individual and later feeds on an uninfected individual.
Fundamentally, disease is a manifestation of interactions between host and pathogen. An array of environmental and physiological factors may influence these interactions. Many aspects of insect behavior and life history are important in disease transmission, especially those relating to relationships between vectors and hosts. Generally, the closer the association between vector and host, the greater the suitability of the vector to transmit the disease.
Aedes aegypti, Aedes albopictus, and Aedes polynesiensis are medically important vectors of pathogens including dengue, yellow fever, filariasis, dog heartworm, West Nile and other encephalites. The transfected strains that have been generated may be used to suppress, eliminate or replace naturally occurring vector populations.
Wolbachia is a genus of obligate, intracellular, maternally inherited bacteria that occur in many insect species. Cytoplasmic incompatibility (CI) is one of several reproductive manipulations caused by Wolbachia. CI occurs in matings between individuals that differ in their Wolbachia infection type and results in early embryonic death. The CI mechanism is unknown as disclosed by Charlat, S., Calmet, C., Mercot, H., 2001 “On the mod resc model and the evolution of Wolbachia compatibility types”, Genetics 159, 1415-1422, (hereinafter Charlat 2001); Poinsot et al. “On the mechanism of Wolbachia-induced cytoplasmic incompatibility: confronting the models with the facts”, Bioessays 25, 259-265, 2003; and Dobson, S. L., Rattanadechekul, W., Marsland, E. J., “Fitness advantage and cytoplasmic incompatibility in Wolbachia single and super-infected Aedes albopictus”, Heredity 93, 135-142, 2004 (hereinafter Dobson 2004). Wolbachia in the male acts to ‘modify’ the sperm, such that karyogamy failure occurs following fertilization, resulting in embryo death. If the female (and resulting fertilized egg) have the same Wolbachia type as her mate, Wolbachia acts to ‘rescue’ the modification, resulting in normal embryo development. Thus, matings between uninfected females and infected males are incompatible, but the reciprocal cross is compatible (unidirectional CI). Unidirectional CI provides Wolbachia-infected females with a reproductive advantage relative to uninfected females, promoting the spread of maternally inherited Wolbachia into uninfected host populations. The ability to spread into host populations has led to the proposed use of Wolbachia in population replacement strategies. Specifically, a desired transgene that is linked to Wolbachia could be ‘seeded’ into a mosquito disease vector population. The Wolbachia infection would then serve as a vehicle, driving the linked transgene into the targeted population. Additionally, the introduced Wolbachia infection may directly have a desired impact on the targeted insect population (i.e., genetic modification of Wolbachia strain not required). Bidirectional CI can occur when two or more Wolbachia types infect the same host population. An example is provided by the parasitoid wasp Nasonia vitripennis. Crosses between N. vitripennis strains that are infected with divergent Wolbachia types (A type or B type) result in incompatibility in both cross directions. Theory predicts that bidirectionally incompatible Wolbachia types cannot persist within a panmictic host population as taught by Rousset, F., Raymond, M., Kjellberg, F., 1991, “Cytoplasmic incompatibilities in the mosquito culex pipiens: how to explain a cytotype polymorphism?”, J. Evol. Biol. 4, 69-81, (hereinafter Rousset 1991); and Dobson, S. L, Fox, C. W., Jiggins F. M., 2002, “The effect of Wolbachia-induced cytoplasmic incompatibility on host population size in natural and manipulated systems”, Proc. R. Soc. London B Biol. Sci. 269, 437-445, (hereinafter Dobson 2002), both incorporated herein by reference. Bidirectional CI causes a ‘battle’ between the Wolbachia types, resulting in the elimination of infections until only one Wolbachia type predominates. The host population is a victim during this battle, as bidirectional incompatibility sterilizes many matings. The CI-induced suppression of the host population is transient however, lasting only until one Wolbachia infection type dominates the host population. Therefore, known examples of bidirectional CI have been either artificially generated or isolated from allopatric populations.
Vector population suppression and elimination strategies are based upon artificially prolonging the bidirectional CI battle as taught by Dobson 2002. In a prior field test of the strategy, releases of bidirectionally incompatible males successfully eliminated a Culex mosquito vector population from a village in Burma (Myanmar) as provided in Laven, H. 1967. “Eradication of Culex pipiens fatigans through cytoplasmic incompatibility”. Nature 216: 383-384. However, the availability of naturally occurring bidirectionally incompatible strains that permitted the Culex strategy remains unique among mosquitoes. Therefore, the use of the suppression/elimination strategy in additional mosquito vector populations requires the ability to artificially generate incompatible strains. Similarly, population replacement strategies also require an ability to generate novel infections. Although the artificial transfer of Wolbachia (transfection) has been successfully accomplished in other insect systems as taught by Boyle et al., “Interspecific and intraspecific horizontal transfer of Wolbachia in Drosophilia” Science 260, 1796-1799, 1993; Sasaki et al., “Interspecific transfer of Wolbachia between two lepidopteran insects expressing cytoplasmic incompatibility; a Wolbachia variant naturally infecting Cadra cautella causes male killing in Ephestia kuehniella”, Genetics 162, 1313-1319, 2002; Hartmann et al., “Trans-species transfer of Wolbachia: micro-injection of Wolbachia from Litomosoides sigmodontis into Acanthocheilonema viteae”, Parasitology 126, 503-511, 2003; and Kang et al., “Superinfection of Laodelphax striatellus with Wolbachia from Drosophila simulans”, Heredity 90, 71-76 2003, prior efforts to generate novel infections in mosquitoes have not proven successful as shown by Sinkins, S. P., O'Neill, S. L., “Wolbachia as a vehicle to modify insect populations.”, In: James, A.M.H.A.A. (Ed.), Insect Transgenesis: Methods and Applications, CRC Press, Boca Raton, Fla., pp. 271-287, 2000, all incorporated herein by reference.
Aedes albopictus (Asian tiger mosquito) is a medically important disease vector of multiple arboviruses and filaria. This mosquito is also an important invasive species, frequently spread by human transport. Since its introduction to the United States, Ae. albopictus has spread to become a leading biting nuisance. Ae. albopictus individuals are naturally co-infected with two Wolbachia types (wAlbA and wAlbB). This type of co-infection is known as ‘superinfection’ and is commonly observed in insects. Superinfection results in additive unidirectional CI: superinfected females express both the A and B rescue and are compatible with all males in the population; superinfected males express both the A and B modification and are compatible only with superinfected females.
Although a majority of Ae. albopictus populations are superinfected, laboratory colonies of single-infected (wAlbA) strains have been established from the islands of Koh Samui and Mauritius. Crosses demonstrate that the superinfection is unidirectionally incompatible with the wAlbA infection.
Crosses of wAlbA-infected females and super-infected males are incompatible, resulting in high embryo mortality. The males in the latter cross differ only by the wAlbB infection present in males.
Aedes aegypti (yellow fever mosquito) is the principle vector of dengue viruses throughout the tropical world. Without a registered vaccine or other prophylactic measures, efforts to reduce cases of dengue fever and dengue hemorrhagic fever are limited to vector control. Unfortunately, traditional mosquito control measures are not succeeding. With an estimated 100 million human cases of dengue fever every year, substantial effect is being devoted to the development of new strategies to complement existing vector control methods.
A gene drive vehicle is an important component of vector population replacement strategies, providing a mechanism for the autonomous spread of desired transgenes into the targeted population. Compared with strategies that rely on inundative releases and Mendelian inheritance, genedrive strategies would require relatively small “seedings” of transgenic individuals into a field population. Perhaps more important than increased cost efficacy, gene drive strategies can facilitate population replacement with transgenic individuals that have a lower fitness relative to the natural population.
As previously noted, Cytoplasmic incompatibility (CI), induced by naturally occurring intracellular Wolbachia bacteria, has attracted scientific attention as a potential vehicle for gene drive. Although CI and other forms of reproductive parasitism have made Wolbachia an evolutionary success, with an estimate that infections occur in ˜20% of insect species, Wolbachia infections do not naturally occur in A. aegypti, raising the questions of whether A. aegypti can support a Wolbachia infection. Key parameters in Wolbachia infection dynamics include the intensity of CI (number of hatching eggs resulting from an incompatible cross), the maternal inheritance rates (number of uninfected progeny produced by an infected female) and mosquito fitness costs associated with the infection. These parameters also determine the infection frequency after a population replacement event, an important consideration because of the goal of population replacement is for the entire mosquito population to carry the desired genotype. The parameters also determine the rate at which the infection will invade the targeted population, an important consideration since the strategy should take place within a “human, not evolutionary, time frame.” (N. Besansky, U. Notre Dame).